In roses, as shown in FIG. 1, the number of hydroxyl groups of ring B (upper right) has a considerable effect on anthocyanidin (flower color), and when the number of hydroxyl groups is three, flower color frequently changes from blue to violet. In fact, many flowers ranging from blue to violet in color contain anthocyanin derived from delphinidin.
Enzymes that govern B ring hydroxylation consist of flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H), and the genes of both enzymes are acquired from numerous plants including petunias. Based on a comparison of amino acid sequences, F3′H and F3′5′H belong sub-families classified into CYP75B and CYP75A, respectively, of the cytochrome P450 super family (refer to Non-Patent Document 1). Recently, enzymes having F3′5′H activity of aster and osteospermum (which are both members of the aster family) have been reported to belong to the CYP75B sub-family. On the other hand, these plants also have enzymes belonging to the CPY75B sub-family that have F3′H activity. Thus, plants belonging to the aster family are presumed to have genes belonging to the CYP75B that overlap, and acquired a function for encoding enzymes having F3′5′H activity (refer to Non-Patent Document 2).
However, a protein designated as F3′5′H of cineraria (Pericallis cruenta or Senecio cruentus), which is also a member of the aster family, is registered as Accession No. AAX1988 of the protein database of the National Center for Biotechnology information. Although examples of measuring the activity of this protein in yeast have been reported (refer to Non-Patent Document 2), since results of measuring the activity of this protein in plants have not been reported, its function is unclear.
Since plants such as roses and carnations are unable to synthesize delphinidin due to the absence of F3′5′H gene, there are no varieties having a violet to blue flower color. It would be industrially useful if it were possible to produce varieties having violet to blue color. Recently, varieties having violet to blue color, which were unable to be obtained with conventional cross-breeding, have been developed by expressing F3′5′H gene in roses and carnations by utilizing genetic recombination techniques (refer to Non-Patent Document 1 and Non-Patent Document 3). In addition, examples have also been reported of changing flower color using genetic recombination techniques (refer to Non-Patent Document 4).
For example, several plants are unable to produce bright red and orange flowers as a result of not producing pelargonidin. Since the dihydroflavonol 4-reductase of flowers such as petunias is unable to reduce dihydrokaempferol, they do not produce pelargonidin. On the other hand, it has been demonstrated experimentally in vitro that inhibiting expression of flavonoid 3′-hydroxylase (F3′H) gene in chrysanthemums results in accumulation of pelargonidin (refer to Non-Patent Document 5). However, there are no chrysanthemums currently known that actually accumulate pelargonidin as a primary anthocyanidin.
In these reports, an exogenous gene is linked to a constitutive promoter or pedal-specific promoter in order to express the exogenous gene in petals. A promoter of a constitutive gene involved in flavonoid biosynthesis is frequently used for the petal-specific promoter. For example, a promoter such as that derived from chalcone synthase present in snapdragons is used in carnations to accumulate delphinidin (refer to Patent Document 1 and Patent Document 2).
However, it is difficult to predict the degree to which a target gene is expressed in a certain plant when using a certain promoter. In addition, a nucleotide sequence required to terminate transcription referred to as a terminator is frequently used in addition to a promoter to express a target gene. Although the gene terminators of nopaline synthase, mannopine synthase and octopine synthase derived from Agrobacterium are frequently used, it is not easy to predict in advance which terminator should be used to allow a target gene to function properly. In addition, although there are also cases in which chromosomal genes of plant genes (translation sequence regions or terminator regions containing promoters and introns) are allowed to function by inserting directly into a plant (refer to, for example, Non-Patent Document 2), in such cases as well, it is difficult to predict whether or not the inserted gene will actually function.
Moreover, plants frequently exhibit a high degree of polyploidy. Cultivated roses are tetraploids, cultivated chrysanthemums are hexaploids, and cineraria are octoploids. Thus, genes of enzymes such as F3′5′H involved in flavonoid synthesis are predicted to be present in these plants in at least the number of the polyploidy. Even if all of these are not transcribed and function, since the plant is able to demonstrate F3′5′H activity, it is not easy to isolate a promoter that is actually able to function from these plants.
A method involving transcription of double-stranded RNA (to also be referred to as RNAi) is generally widely used to inhibit gene expression.
In red petunias that produce cyanidin, expression of F3′H gene is inhibited by transcribing its double-stranded RNA, while at the same time, petunias having an orange color are obtained by causing pelargonidin to accumulate by expressing DFR enzyme gene derived from roses (refer to Non-Patent Document 6).
In addition, in chrysanthemums as well, there are examples of inhibiting expression of F3′H gene, causing excess expression of pansy F3′5′H gene and accumulating delphinidin by transcribing its double-stranded RNA (pCGP3429, refer to Patent Document 3).
When transcribing double-stranded RNA, a cDNA-derived sequence (such as an F3′H cDNA sequence) or an unrelated sequence (such as an E. coli-derived GUS sequence) can be inserted between sequences generating an inverted repeat sequence. Although inhibition efficiency of a target gene has been reported to be increased when an intron sequence is inserted therein (refer to Non-Patent Document 7), since there are numerous types of intron sequences, it is not easy even for a person skilled in the art to predict which specific intron sequence should be used.